The present invention concerns solid compositions and methods useful in separating chemical components in a complex mixture in which at least one of the chemical components is capable of being selectively adsorbed. The invention also concerns methods of reducing non-specific binding of the other components in the mixture.
Separations of components in a mixture have been important in many scientific disciplines, such as chemistry, biochemistry and molecular biology. The separation of components in a mixture allows isolation of the component of interest, i.e., an analyte. After the analyte is isolated, the properties of the analyte can be studied or used. Without the separation, it may be difficult to determine the properties of the analyte because, whatever the measurement technique used, the properties of the component of interest could be masked or influenced by other components in the mixture. Thus, separation techniques can be considered as corner stones in scientific studies.
The separation of components is made difficult if the mixture containing the analyte is a complex mixture. Good examples of complex mixtures include media of biological fermentation, cell cultures, transgenically produced milk, or slurries of transgenic plant matter, in which a specific analyte is desired and needs to be separated and purified. The separation of components in the complex mixture is usually accomplished by affinity separation techniques. The affinity separation technique usually involves contacting the mixture with a solid phase having a functionality specifically designed to bind to the analyte, but be substantially non-reactive with other components in the mixture, thereby leaving the other components free to be removed. After the non-bound components are removed, e.g. by washing the solid phase with water or buffer, the analyte is left behind bound to the solid phase, so the analyte is separated from the non-bound components. The analyte is then isolated by separating the analyte from the solid phase, usually by a buffer change, to recover the analyte as free molecules.
Classes of valuable xe2x80x9caffinityxe2x80x9d techniques for purification have been developed. These techniques have many names, affinity chromatography, affinity precipitation, immunoaffinity separation, etc., but they all rely on the same principles, that is, a specific functionality or binding moiety is chemically attached to a solid support that binds very selectively to the target analyte. The most common binding moieties for protein purification are other proteins such as Protein A or Protein G, or monoclonal antibodies, chelated metals ions, polypeptides, or small organic molecules. Monoclonal antibodies can be especially attractive for protein purification because they can be highly selective for the target protein. As indicated above, the mixture that contains the analyte is allowed to contact the affinity solid support with the binding moiety attached. The analyte binds to the binding moiety on the support and the rest of the mixture is removed. The analyte is then removed from the binding moiety by elution, usually achieved by changing the solvent. Very high purification factors can be realized. There is extensive literature on affinity techniques1-8.
Recent developments in the selection and production of monoclonal antibodies have made the affinity technique based on the monoclonal antibody as the binding moiety a very powerful technique for the purification of proteins and biopharmaceuticals. Monoclonal antibodies are proteins themselves that are often purified from cell culture or fermentation using affinity purification that uses Protein A or Protein G as the ligand. New small organic Protein A mimetics have also been described as useful ligands for monclonal antibody purification.
Although affinity purification has proven to be a powerful technique, its full potential has not been fully realized. It is most commonly practiced where the support is formed into small beads, on the order of 0.05 to 0.5 mm or so, and the beads, often referred to as media, are loaded into a chromatography column. The mixture to be purified is then passed through the column and the analyte binds to the binding moiety attached to the media. The column is then washed extensively to remove the occluded mixture. An elution solvent is then passed through the column liberating the analyte in solution. On a large-scale, this process requires that the media have good physical strength to handle the weight and turbulence encountered in column applications.
Certain supports currently used in affinity separations, whether as column chromatography or some other system, are low surface area materials, such as carbohydrate-based materials or polymers. These low surface area supports can have low capacity. Because of the low capacity, relatively large loadings of media are needed to recover the target species. But, with large loadings of media, flow rates over the column are restricted to low rates due to pressure drop considerations. Column chromatography can also be practiced under high pressure where smaller beads are used to increase the capacity of the media. Because these beads must have higher strength to handle the pressure, carbohydrate gels are cross-linked, thereby lowering the capacity of the resulting beads. Therefore, there is a need to provide affinity supports with high capacity and which are further physically robust when used in high pressure liquid chromatography.
Developing high surface area supports is one approach to obtaining high capacity affinity separation media. With a higher capacity material, smaller amounts of the affinity support is needed to recover the target species, column pressure drops are lower, flow rates are higher, and there is less occluded feed contamination. High surface areas could range from 10-500 m2/g. Materials that can provide high surface area are silica gels, silicas, aluminas, zirconias, carbohydrates, and polymeric materials such as macropore acrylic beads. In the case of silica gels, surface areas can vary from very low, 1 m2/g, to very high, in excess of 800 m2/g, with pore size modes from very low, less that 25 xc3x85 to in excess of 1500 xc3x85. Furthermore, inorganic oxide-based materials are usually much more physically robust than the softer carbohydrate based supports.
When used as media in affinity separation techniques with a binding moiety attached, these oxide based materials, while having the requisite high surface area, can suffer from a high degree of non-selective binding of unwanted materials. Not all of the surface area will be used for the affinity separation; some will actually provide surface regions for non-selective adsorption. It is well known that proteins bind very strongly to silica for instance, sometimes irreversibly and non-selectively. Therefore, while the binding moiety can be very selective, the unused regions of the surface will be non-selective. The net effect is to lower the selectivity of the high surface area materials, thereby reducing the purification factors of the overall process. This non-selective adsorption by many oxide supports, and especially silicas such as silica gels, is the reason these materials are currently not used extensively as affinity separation supports.
One of the objectives of this invention to describe a surface composition to be applied to high surface area materials which improve the non-selective adsorption while retaining the high capacity for the selective affinity binding.
Such compositions will have great value in xe2x80x9caffinity separationsxe2x80x9d from complex biological mixtures where specific biological species, such as proteins, are synthesized by genetically engineered organisms. For instance the complex mixture might be a fermentation broth for cellular or bacterial production of a target protein. The fermentation broths are complex mixtures of proteins, carbohydrates, etc., that support the organism growth, as well as by products produced by the fermentation. The target species can also be produced from the fermentation and is produced by the organism into the broth. In some cases, the target species is produced in the cell. Recovery is therefore complicated by the fact the cells need to be homogenized and the target dissolved. These mixtures are particularly insidious for target species isolation and purification. Separation and purification schemes for the isolation and purification of the target species from fermentation broths are very complicated and expensive. The cost of isolation and purification is especially significant as the large-scale production. Because of the challenging nature of this problem, the field of purification and isolation is extensive.
The solid composition of the invention comprises an inorganic substance and moiety R10 located on at least one surface of said inorganic substance, wherein
said inorganic substance is an inorganic oxide
and said R10 group is an entity selected from the group consisting of xe2x80x94CH2OH, xe2x80x94CH(OH)2, xe2x80x94CH(OH)CH3, xe2x80x94CH2CH2OH, xe2x80x94C(OH)2CH3, xe2x80x94CH2CH(OH)2 and xe2x80x94CH(OH)CH2(OH).
When R10 is an entity selected from the group consisting of xe2x80x94CH2OH, xe2x80x94CH(OH)2, xe2x80x94CH(OH)CH3, xe2x80x94CH2CH2OH, xe2x80x94C(OH)2CH3, xe2x80x94CH2CH(OH)2 and xe2x80x94CH(OH)CH2(OH), the solid support possesses a distinctive characteristic of having reduced non-specific binding of any non-analyte components in a complex mixture. The members of R10 have a common property of having zero electric charge and being hydrophilic. Without being held to any particular theory, it is believed that when the solid support has any of the R10 entities, i.e. xe2x80x94CH2OH, xe2x80x94CH(OH)2, xe2x80x94CH(OH)CH3, xe2x80x94CH2CH2OH, xe2x80x94C(OH)2CH3, xe2x80x94CH2CH(OH)2 and xe2x80x94CH(OH)CH2(OH), located on its surface, the binding of the non-analyte component in the mixture to the surface has an entropy change lower than the remaining non-analyte component in the mixture""s aqueous phase. Binding of any component, e.g., non-analyte or analyte component, from a solution to a surface involves a lowering of entropy due to localization of the non-analyte on the surface. In order for binding to be favorable, there has to be an interaction between non-analyte and surface, such as a Columbic charge interaction or hydrophobic interaction, to overcome the lowering of entropy due to surface localization. Coating with any one of or any mixture of the entities for R10, however, produces a hydrophilic surface, as well as a surface having a zero net charge which reduces interaction necessary for reducing entropy and accordingly reduces non-binding of non-analyte.
Also within the scope of the present invention are solids comprising the inorganic substance, moiety R10 located on at least one surface of the inorganic substance and at least one binding moiety capable of binding analyte. The moiety R10 is preferably located on a portion of the inorganic oxide""s surface and on the remainder of the surface is located binding moiety. The binding moiety is selected to provide bonding to a particular analyte and provides the highly selective binding for affinity separations, while the surface coated with R10 groups reduces non-selective binding of other species. In preferred embodiments, a significant, if not a large majority, of the inorganic oxide surface is covered by R10 groups, e.g., at concentrations of about 1 to 10 groups per nm2 substrate, and the remainder covered by the binding moiety.
Also within the scope of the invention is a solid comprising the inorganic substance in which binding moiety is optionally located on the inorganic oxide surface through a linker. The linker can be located on the inorganic oxide surface by reacting a linker compound with the inorganic oxide and then subsequently reacting the linker with the binding moiety. Accordingly, the invention contemplates inorganic substances comprising linker groups and R10 moieties located on its surface. This solid in turn can be transferred to an end user who can attach a specifically designed binding moiety to the linker for use in a separation.